There is widespread agreement that chronic alcohol exposure leads to mitochondrial damage. An unexplored aspect of mitochondrial homeostasis is the mitochondrial stress response (MSR) which regulates matrix chaperone production (e.g., Hsp60 and 10) and mitochondrial biogenesis in response to stress to mitochondria (e.g., protein malfolding or oxidative modifications) as would occur from alcohol. The MSR and biogenesis responses are mediated mainly by up-regulation of nuclear genes. We have begun to explore the upstream sensor and trigger of these responses, referred to as mitochondrial retrograde signaling and our Preliminary Results have identified a key transcriptional co-activator (TORC3) which resides in the mitochondrial matrix and appears to translocate to the nucleus after mitochondrial damage. Therefore, the aims of the current application are to: 1) Confirm localization of TORC3 in mitochondria and compare with localization of TORC2. TORC 2 and 3 are two transcriptional co-activators in liver which up-regulate Hsp60 and PGC-1a. The hypothesis that there is separate compartmentation of TORC2 (cytosol) and TORC3 (mitochondria matrix) will be carefully tested defined using differential or density gradient centrifugation, mitoplasts and high resolution confocal imaging: 2) Examine the role of TORC3 in MSR and mitochondrial biogenesis in cell culture models (HepG2 cells and primary mouse hepatocytes) of mitochondrial damage using rotenone, antimycin A, or expression of Cyp2e1 in mitochondria with the goals of (a) characterizing the detailed time course of TORC3 release from mitochondria and nuclear translocation in relation to MSR and biogenesis gene expression in response to mitochondrial oxidative stress (b) comparing nuclear translocation of TORC2 versus TORC3 to Hsp60 promoter and the effect of silencing TORC2 and or TORC3 on MSR and mitochondrial biogenesis as well as susceptibility to lethal mitochondrial injury; (c) determining the role of CHOP and concomitant ER stress in relation to TORC2 and TORC3 on mitochondrial stress and biogenesis responses; these studies will utilize real-time PCR, Western blots, ChIP assays and siRNA; 3) Define the effect of pyrazole-induced Cyp2e1 on MSR and mitochondrial biogenesis in vivo; preliminary results confirm pyrazole induces mitochondrial and ER Cyp2e1 and this causes MSR; therefore, pyrazole treatment may provide a convenient in vivo model for selective mitochondrial damage; 4) Describe the expression pattern of MSR and mitochondrial biogenesis genes in chronic intragastric alcohol fed mice. The results of this application should convincingly characterize a novel direct retrograde signaling pathway from mitochondria to nucleus involving TORC3 transcription co-activator, define its role relative to other signaling pathways, and begin to establish its relevance to alcohol injury in vivo. [unreadable] [unreadable] [unreadable]